This proposal is to perform a comparative study of the secreted chemicals and receptors that regulate behavior in nine species of nematodes from two genera. Using recent knowledge of several molecules that cause mating behavior or dauer formation in the model organism Caenorhabditis elegans, new compounds will be identified and characterized from eight additional Rhabditid species: C. briggsae, C. remanei, C. brenneri, C. japonica, Pristionchus pacificus, P. lheritieri, P. uniformis, and P. pauli. This study will expand our knowledge of chemical signaling and provide an important biological foundation for the chemical control of behavior in these nematodes that have a variety of ecological niches and reproductive modes. The work will be accomplished by isolating worm-conditioned water at defined developmental stages. This worm water will be essentially free from bacterial compounds and will be used in both targeted and activity-guided structure determination using NMR and mass spectrometry. The targeted analysis will focus on compounds from all species that contain the dideoxysugar ascarylose, which is attached to different fatty-acid derived groups in all known C. elegans pheromones. In the activity-guided identification, worm conditioned water will be fractionated and bioassayed for both dauer and mating activities. Compounds identified through the targeted aim will be fractionated in identical protocols and dereplicated so that only unique compounds will be studied in the activity-guided aim. Novel compounds from both aims will be synthesized and tested for biological activity, both in the source species and between all species. This aim will not only provide detailed information about mating and dauer formation in each individual species but also will provide the basis to better understand the ecology and behavior of rhabditid nematodes. Finally, with the known compounds for both dauer and mating activities, receptors for these molecules will be identified through genetic screens and profiling cDNAs in cells that are necessary for mating activity. Knowledge of the receptors will help connect chemistry and behavior with the extensive existing knowledge of genetics of dauer and mating pathways in C. elegans. Relevance: Nematodes are the most abundant animal on earth, and parasitic species infect billions of humans, other animals including domestic pets and farm stock, and plants including major agricultural food sources. Knowledge gained from this proposal might provide new starting points for drug discovery to control parasitic nematodes that adversely impact the health of billions of people in the world. PUBLIC HEALTH RELEVANCE: Nematodes are the most abundant animal on earth, and parasitic species infect billions of humans, other animals including domestic pets and farm stock, and plants including major agricultural food sources. Knowledge gained from this proposal might provide new starting points for drug discovery to control parasitic nematodes that adversely impact the health of billions of people in the world.